Erythromycin e

ABSTRACT

Erythromycin E is useful as an antibiotic. The compound is prepared by the fermentation of erythromycin with Streptomyces erythreus NRRL 3887 in suitable nutrient media.

United States Patent [191 Martin et a].

[451 Apr. 2, 1974 ERYTHROMYCIN E [75] Inventors: Jerry Roy Martin,Waukegan; Alma W. Goldstein, Lake Bluff, both of 111.

[73] Assignee: Abbott Laboratories, North Chicago, Ill.

[22] Filed: Jan. 22, 1973 [21] Appl. No.2 325,695

Related US. Application Data [62] Division of Ser. No. 107,428, Jan. 18,1971, Pat. No.

[52] US. Cl 195/80 R I51] Int. Cl Cl2d 9/00 [58] Field of Search 195/80R [56] References Cited UNITED STATES PATENTS 3,714,172 l/l973 Martin eta]. 195/80 R X [57] ABSTRACT Erythromycin E is useful as an antibiotic.The compound is prepared by the fermentation of erythromy cin withStreptomyces erythreus NRRL 3887 in suitable nutrient media.

1 Claim, No Drawings IERYTHROMYCIN E This compound is prepared by thefermentation of erythromycin in a nutrient media comprising acarbohydrate energy source such as a monosaccharide, e.g., glucose andcorn starch; and a source of nitrogen such as soy flour, corn steepliquor, ammonium nitrate and the like, which media has been seeded witha culture of Streptomyces erythreus NRRL 3887. It is preferred that thenutrient media comprise a buffering agent to moderate the pH as thefermentation progresses. Such buff- 8.5. A filter aid, Dicalite, wasadded and the mixture was stirred for 5 minutes. The mixture wasfiltered and the clear filtrate was collected. The filtrate wasextracted twice with '16 volume of ethyl acetate. The combined ethylacetate extract was washed two times with water and dried over anhydrousM gSO Concentration in vacuo gave 1.1 12 g. of dark viscous oil. The oilwas added to the top of a column of Sephadex 1.1-1-20 (2.2 X 90 cm)prepared and eluted with methanol. Eluded fractions monitored by thinlayer chromatography, showed three major components only partiallyseparated from one another. Thin layer chromatographic comparisonindicated that two of the components were known compounds: unchangedadded erythromycin A and anhydroerythromycin A. Fractions enriched inthe third component were pooled and concentrated to dryness to give 448mg. of pale yellow oil. This material was chromatographed on a column ofsilica gel according to the method of Oleinick and Corcoran Jour. Biol.Chem. 244:727( 1969). The composition of each fraction was determined bythin layer chromatography. Fractions containing only the unknownmaterial were pooled and concentrated in vacuo to give 98.7 mg. of

ering agents which are suitable include dihydrogen phosphate andalkaline earth carbonates, e.g., calcium carbonate. The foregoingdescription of the compound of this invention will now further beillustrated by a specific example setting forth the best mode ofpreparing thecompound.

Seed cultures of Streptomyces erythreus NRRL 3887 were prepared in amedium consisting of (in grams per liter) glucose monohydrate(Cerelose),15.0; soybean meal, 15.0; and CaCO 1.0. The cultures were incubated at32 C. for 72 hours on a rotary shaker. The seed was added at a level of35% (v/v) into 500 ml. Erlenmyer flasks containing 50 ml. of afermentation medium consisting of the following components in grams:

Corn starch 15.0 Soybean meal 20.0 Corn steep 50.0 CaCO, 1.0 Soybean oil(Edsoy) 50.0 Tap water 1000 The medium was adjusted to pH 6.8 withsodium carbonate prior to sterilization. The fermentation flasks wereincubated at 32 C. in a rotary shaker (250 rpm) for 48 hours, then 25mg. of the finely divided erythromycin A was added to each of flasks.Incubation with shaking was continued for an additional 120 hours, thenthe flask contents were pooled and clarified as follows. To thefermentation contents was added, with stirring, an equal volume of anaqueous solution of 10% zinc sulfate followed by a volume of 0.5 Nsodium hydroxide sufficient to raise the of the solution to white solid.Phosphate salts were removed from the preparation by passage through acolumn of Sephadex LH-20 prepared in methanol. The salt free solids wereconcentrated in vacuo to give a colorless oil. Crystallization fromether-hexane gave 33 mg. of colorless prisms softening at and melting atAs the following experimental results illustrate, erythromycin E has aspectrum of activity similar in character to erythromycin A. In thetable there is shown the activity of erythromycin E against a number ofbacterial strains and certain other microorganisms.

In addition to erythromycin A, as disclosed in the above example,erythromycin B and C can also be used as starting material in thedescribed fermentation with good results.

TABLE Minimum Inhibitor Concentration in Micro-organism meg/m1 at pH 7.4

Pharmaceutically acceptable non-toxic salts include single entities andmixtures of these acid addition salts which include the hydrochloride,the hydrobromide, the hydroiodide, lactobionate, thiocyanate, both loweralkyl sulfates and the higher alkyl sulfates, such as stearyl sulfate,lauryl sulfate, and cetyl sulfate, alkyl and aryl sulfonates,phosphates, sulfates, maleates, fumarates, succinates, tartrates,citrates, stearates, and others commonly used in the art.

Salts obtained through variation of the acid used to neutralize the basecompound and form the acid addition salt have special advantages in someinstances because of their increased stability, increased solubility,decreased solubility, ease of crystallization, or lack of objectionabletaste. Such advantages accrue because of the ease of administration andassimilation of the particular acid addition salt and such propertiesare subsidiary to the main physiological action of theindividneutralization reaction and can be carried out in any suitablesolvent through the addition of a chemical equivalent of the base.

The compound of this invention may be administered orally orparenterally in conventional dosage forms such as tablets, capsules,injectables and the like, which incorporate erythromycin E alone, andwith other pharmacodynamically active substances or with suitablecarriers according to accepted pharmaceutical practices. To obtainantibiotic responses against susceptible organisms, the desired dosagerange is from about to 200 mg/kg of body weight.

I claim:

1. A process for preparing erythromycin E comprising culturingstreptomyces erythreus NRRL 3887 in a nutrient medium comprising acarbohydrate source, a nitrogen source and added erthromycin selectedfrom the group consisting of erthromycin A, B, and C.

